The present invention relates to a modified protein which has a liver accumulation property that its binding affinity for an asialoglycoprotein receptor present on a surface of a hepatocyte is lower than that of an asialoorosomucoid, and which can be produced by reacting a physiologically active protein containing one Gln residue that becomes a substrate for a transglutaminase with a branched-chain ligand composed of an amino acid derivative containing branched Gal or branched GalNAc in the presence of a transglutaminase to form an amide bond between the xcex3-carboxyamide group of the glutamine residue in the physiologically active protein and the terminal primary amino group in the branched-chain ligand, a process for producing the same, and a pharmaceutical composition containing the above-mentioned protein.
The modified protein of the present invention is useful as an active ingredient of medications since the physiologically active protein used as a starting material retains a physiological activity.
In recent years, as a result of the development of the biotechnology, a variety of physiologically active proteins can be mass-produced, and they have been expected to be candidate substances of new medications. However, there have been a large number of problems to be solved in order to put the same into practical use. Of these, the strict control of the pharmacokinetics thereof has been considered to be an important subject for increasing therapeutic effects and reducing side effects. For example, a great many investigations have been conducted on antitumor effects of recombinant human interleukin-2 (rhIL-2). The effects are identified with respect to mouse sarcoma and mammary tumor, and antitumor effects to melanoma and hemangioendothelioma are clinically identified. However, no expected effects to solid carcinoma of digestive organs are identified with rhIL-2 alone either in the animal test or clinically. Further, since rhIL-2 has a short half-life in the blood after the intravenous administration, a high dose thereof is required to exhibit the antitumor activity. Nevertheless, when rhIL-2 is administered at a high dose, a serious side effect called capillary leak syndrome occurs, providing an. influence such as an edema or the like in the lung or the liver. It is necessary to control the chemotherapeutic fate thereof for increasing the therapeutic effect of rhIL-2. In order to solve such a problem, investigations have been lately conducted with respect to an immunotherapy on metastatic and primary liver cancers by accumulating rhIL-2 around liver sinusoidal cells using IL-2 preparations of rhIL-2-containing liposomes or galactose-containing liposomes and increasing an activity of liver sinusoidal lymphocytes or the like (Jpn. J. Cancer Chemother., (1994), 21(13), 2105-2107).
Meanwhile, it is known that mammallian hepatocytes have an asialoglycoprotein receptor (hereinafter abbreviated as xe2x80x9cASGRxe2x80x9d) which is a specific membrane-binding receptor to a glycoprotein having galactose (hereinafter abbreviated as xe2x80x9cGalxe2x80x9d) or N-acetylgalactosamine (hereinafter abbreviated as xe2x80x9cGalNAcxe2x80x9d) in a branched sugar chain terminal (Ashwell, G., et al., Annu. Rev. Biochem., 1982, 51, 531-554). An uptake mechanism of the sugar protein with the receptor has a high binding affinity and is strong. In view of these properties and the specific presence of ASGR in hepatocytes, the above-mentioned receptor has attracted attention as a targeting system for specifically delivering medicines or genetic DNAs in metabolically important target cells (Wu, G.
Y. et al., J. Biol. Chem., (1987), 262, 4429-4432).
As a result of studies on the structure-activity interrelation of branched oligosaccharide chains or synthetic Gal derivatives isolated in the sugar recognition mechanism with the above-mentioned receptor, it has been clarified in view of the distance between Gal residues and the branching pattern that the intensity of the binding affinity is in the order of tetra-antenna type Gal greater than tri-antenna type Gal greater than bi-antenna type Gal greater than mono-antenna type Gal (Lee, Y. C. et al., J. Biol. Chem., (1983), 258, 199-202; Kawaguchi, K. et al., Arch. Biochem. Biophys., (1980), 205, 388-395; Connolly D. T. et al., J. Biol. Chem., (1982), 257, 939-945; Lee, R. T., et al., Biochemistry, (1984), 23, 4255-4259).
According to these findings, attempts have been made to accumulate DNAs or liposomes in the liver using a synthetic ligand containing branched Gal or GalNAc and to incorporate the same into cells (Japanese Laid-Open (Kokai) No. 202,085/1993; Haensler J. et al., Bioconjugate Chem., (1993), 4, 85-93; and Merwin, J. R., Bioconjugate Chem., (1994), 5, 612-620).
However, in the drug delivery system (hereinafter abbreviated as xe2x80x9cDDSxe2x80x9d) using an endocytosis mechanism through such a receptor, a low efficiency of intracellular uptake into the cells through a synthetic ligand is at issue. It has been made clear that this is because the binding affinity of such a synthetic ligand for ASGR is lower than a sugar chain of a glycoprotein such as asialoorosomucoid (ASOR), asialofetuin or the like which is a natural ligand [Lee, R. T. et al., Glycoconjugate J., (1987) 4, 317-328; and Biessen, E. A. L. et al., J. Med. Chem., 1995, 38, 1538-1546). Because of the low binding affinity, the synthetic ligand having Gal or GalNAc cannot be bound well to ASGR, and it is hard to incorporate ASGR into hepatocytes. Thus, the pharmaceutical effect is not exhibited well.
On the other hand, we have developed a method in which a protein is site-specifically modified with alkylamine derivatives of various compounds using an animal transglutaminase. Nevertheless, in this method, it is difficult that alkylamine derivatives are bound to a glutamine residue in an amino acid sequence of a physiologically active protein, for example, rhIL-2. Thus, a modified compound in which the alkylamine derivative is bound to the physiologically active protein derivative by introducing a peptide having a glutamine residue which is bindable using an animal transglutaminase into the physiologically active protein (PCT/JP 95/00298 (WO 96/06181)).
We have further investigated that the glutamine residue in the amino acid sequence of the physiologically active protein can be site-specifically modified using, among transglutaminases, especially a transglutaminase (B-TG) derived from microorganisms and having a wide substrate specificity to a Gln residue of a protein and using polylysine or alkylamine derivatives of an polyalkylene glycol such as polyethylene glycol without newly introducing a peptide molecule into an amino acid sequence of a physiologically active protein such as rhIL-2 or the like (Japanese Patent Application No. 270,102/1994 (Japanese Laid-Open (Kokai) No. 89,278/1996)); and PCT/JP 95/01994 (WO 96/10089)).
The present inventors have conducted various investigations to solve the problems that the compound modified with the synthetic ligand has a low affinity for ASGR and the modified compound is hardly incorporated into hepatocytes, and that for producing such a compound modified with the synthetic ligand, a peptide having a Gln residue has to be bound to a physiologically active protein.
The present inventors have attracted attention to the fact that if the physiologically active protein modified with such a synthetic ligand can directly act on cells other than hepatocytes, for examples, liver sinusoidal lymphocytes to exhibit the pharmaceutical effect of the physiologically active protein, the physiologically active proteins such as cytokine and the like are accumulated in the liver rather utilizing the low binding affinity for hepatocytes provided by such a synthetic ligand-modified compound, and that since the physiologically active substance is hardly incorporated into hepatocytes, it can be maintained in the liver, with the result that the activity of cytokine is expected to be exhibited by being bound to a specific receptor present on a cell membrane of the other target cell in the liver.
The targeting to the liver results in decreasing the amount of the protein to be shifted to the lung or the like, making it possible also to decrease the side effect by rhIL-2 in organs other than the target organ.
And it has been considered that in order to effectively exhibit the pharmaceutical effect of the physiologically active protein having such a liver accumulation property upon increasing the activity of the liver sinusoidal lymphocytes without incorporating the same into hepatocytes, this physiologically active protein has to be modified directly while maintaining the activity of the physiologically active protein as much as possible without formulating the physiologically active protein into preparations using a liposome or the like.
For example, attention has been drawn to the fact that if it is possible that rhIL-2 is site-specifically modified with a branched synthetic ligand such as Gal or GalNAc while maintaining the activity of rhIL-2 and is accumulated around hepatocytes without being incorporated into hepatocytes, it is bound to the IL-2 receptor (IL-2R) on liver sinusoidal lymphocytes as target cells whereby the activity is enhanced and antitumor effects to metastatic and primary liver cancers are expected.
However, the knowledges over the liver accumulation of the protein modified with such a branched ligand and the development of the activity thereof in the liver have not yet been obtained. Needless to say, the knowledge over the decrease in the side effect in organs other than the target organ has not yet been obtained at all.
The present inventors have found that recombinant human interleukin-2 (rhIL-2) site-specifically modified with a branched Gal ligand having an acidic or basic amino acid structure using a transglutaminase (B-TG) derived from microorganisms retains a biological activity compared to unmodified one, exhibits a liver accumulation property, is hardly incorporated into hepatocytes, and further minimizes remarkably the tumor in the mouse liver tumor model. Likewise, it has been found that human interferon-xcex1modified with a branched Gal ligand retains a biological activity compared to unmodified one, and exhibits a liver accumulation property.
It has been further found that an IL-2 fusion protein obtained by introducing one molecule of the Gal ligand and one molecule of the polyethylene glycol derivative site-specifically using two types of transglutaminases exhibits a higher liver accumulation property.
The present invention has been completed on the basis of such findings.
That is, it is an object of the present invention to provide a modified physiologically active protein which is obtained by modifying a protein that is expected to exhibit a physiological activity in the liver with a synthetic ligand upon using a lower binding affinity of the synthetic ligand for ASGR than that of a natural sugar chain for ASGR, whereby the physiologically active protein is selectively accumulated in the liver, target cells to the physiologically active protein present in the liver are activated, or active oxygen formed is removed to increase the antitumor effect, the antiviral effect and the anti-inflammatory effect, and the amount of the modified physiologically active protein delivered into the blood and other organs is decreased to reduce the side effect, as well as a pharmaceutical composition containing the same and having the excellent liver accumulation property.
Another object of the present invention is to provide a pharmaceutical composition containing the above-mentioned modified physiologically active protein and a pharmaceutically acceptable carrier, this composition having a liver accumulation property and reducing a side effect.
Still another object of the present invention is to provide a process for producing a modified physiologically active protein by modifying site-specifically a physiologically active protein using a transglutaminase derived from microorganisms.
The present invention relates to a pharmaceutical composition having a liver accumulation property, comprising a modified physiologically active protein which can be produced by reacting a physiologically active protein containing at least one Gln residue that becomes a substrate for a transglutaminase and having a molecular weight of from 1xc3x97103 to 2xc3x97105 with a branched-chain ligand composed of an amino acid derivative containing an amino group and a galactose (Gal) group or an N-acetylgalactosamine (GalNAc) group that become substrates for a transglutaminase, the branched-chain ligand having a lower binding affinity for an asialoglycoprotein receptor present on a surface of a hepatocyte than an asialoorosomucoid, in the presence of a transglutaminase derived from microorganisms to form an amide bond between the xcex3-carboxyamide group of the glutamine residue in the physiologically active protein and the terminal amino group in the branched-chain ligand; and a pharmaceutically acceptable carrier.
Further, the present invention relates to a modified physiologically active protein which has a liver accumulation property that its binding affinity for and asialoglycoprotein receptor present on a surface of a hepatocyte is lower than that of an asialoorosomucoid, and which can be produced by reacting a physiologically active protein containing at least one Gln residue that becomes a substrate for a transglutaminase and having a molecular weight of from 1xc3x97103 to 2xc3x97105 with a branched-chain ligand composed of an amino acid derivative containing an amino group and a galactose (Gal) group or an N-acetylgalactosamine (GalNAc) that become substrates for a transglutaminase, the branched-chain ligand having a lower binding affinity for an asialoglycoprotein receptor present on a surface of a hepatocyte than an asialoorosomucoid, in the presence of a transglutaminase derived from microorganisms to form an amide bond between the xcex3-carboxyamide group of the glutamine residue in the physiologically active protein and the terminal amino group in the branched-chain ligand.
Still further, the present invention relates to a process for producing the modified physiologically active protein, which comprises reacting a physiologically active protein containing at least one Gln residue that becomes a substrate of a transglutaminase and having a molecular weight of from 1xc3x97103 to 2xc3x97105 with a branched-chain ligand composed of an amino acid derivative containing an amino group and a galactose (Gal) group or an N-acetylgalactosamine (GalNAc) group that become substrates of a transglutaminase, the branched-chain ligand having a lower binding affinity for an asialoglycoprotein receptor present on a surface of a hepatocyte than an asialoorosomucoid, in the presence of a transglutaminase derived from microorganisms to form an amide bond between the xcex3-carboxyamide group of the glutamine residue in the physiologically active protein and the terminal amino group in the branched-chain ligand.